Vol. 57, No. 6APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1829-18340099-2240/91/061829-06$02.00/0Copyright © 1991, American Society for Microbiology

Purification and Amino Acid Sequence of Lactocin S, a BacteriocinProduced by Lactobacillus sake L45

C. I. M0RTVEDT,l J. NISSEN-MEYER,2 K. SLETTEN,3 AND I. F. NES2*Norwegian Food Research Institute, N-1430 As,1 Laboratory of Microbial Gene Technology, N-1432 As-NLH,2 and

Department of Biochemistry, University of Oslo,3 Oslo, Norway

Received 31 December 1990/Accepted 10 April 1991

Lactocin S, a bacteriocin produced by Lactobacillus sake L45, has been purified to homogeneity by ionexchange, hydrophobic interaction and reverse-phase chromatography, and gel filtration. The purificationresulted in approximately a 40,000-fold increase in the specific activity of lactocin S and enabled thedetermination of a major part of the amino acid sequence. Judging from the amino acid composition, lactocinS contained approximately 33 amino acid residues, of which about 50% were the nonpolar amino acids alanine,valine, and leucine. Amino acids were not detected upon direct N-terminal sequencing, indicating that theN-terminal amino acid was blocked. By cyanogen bromide cleavage at an internal methionine, the sequence ofthe 25 amino acids (including the methionine at the cleavage site) in the C-terminal part of the molecule was

determined. The sequence was Met-Glu-Leu-Leu-Pro-Thr-Ala-Ala-Val-Leu-Tyr-Xaa-Asp-Val-Ala-Gly-Xaa-Phe-Lys-Tyr-Xaa-Ala-Lys-His-His, where Xaa represents unidentified residues. It is likely that the unidentifiedresidues are modified forms of cysteine or amino acids associated with cysteine, since two cysteic acids per

lactocin S molecule were found upon performic acid oxidation of lactocin S. The sequence was unique whencompared to the SWISS-PROT data bank.

Lactic acid bacteria produce a number of antimicrobialsubstances such as lactic acid, H202, diacetyl, and bacteri-ocins (18). Bacteriocins are proteinaceous compounds thatact bactericidally against closely related species. They maybe produced by both gram-positive and gram-negative or-ganisms (3, 32). Bacteriocin-producing lactic acid bacteriamay prove to be useful in improving the safety of feed andfood fermentation products. Today only one bacteriocinfrom lactic acid bacteria, nisin, has found practical applica-tion. In the early 1950s, Hirsch et al. (12) suggested the useof inhibitory streptococci for food preservation. Nisin-pro-ducing starter cultures were successfully used to prevent gasproduction of clostridia in cheese (23). Various applicationsof nisin in the production of food commodities have sincebeen developed. Although a great number of reports on theexistence of new bacteriocins have been published, verylittle information on their chemical composition, mode ofaction, and genetics has emerged. The molecular structure ofnisin, however, has been determined by Gross and Morell(10). Nisin was shown to be a 3,500-Da peptide consisting ofunusual amino acids, and the gene encoding this bacteriocinwas recently cloned (4, 9, 16).

Within the genus Lactobacillus, a number of bacteriocin-ogenic substances have been reported in L. acidophilus (1,26), L. helveticus (14, 33), L. fermenti (7), L. plantarum (6),and L. sake (24, 30). Only a few of these substances havebeen partially purified and characterized. From thermophiliclactobacilli, however, genes of two bacteriocins have beencloned and sequenced: helveticin J from L. helveticus (15)and lactacin F from L. acidophilus (28).M0rtvedt and Nes (24, 25) have described an L. sake-

produced bacteriocin, termed lactocin S, which inhibitsgrowth of selected species of the genera Lactobacillus,Pediococcus, and Leuconostoc. The lactocin S productionand its immunity factor were shown to be associated with an

* Corresponding author.

unstable 50-kb plasmid (25). This study describes for the firsttime a procedure for purifying to homogeneity a bacteriocinfrom a facultative heterofermentative Lactobacillus sp. anddetermination of its amino acid composition. Approximately75% of the amino acid sequence of lactocin S was deter-mined.

MATERIALS AND METHODS

Bacterial cultures and media. L. sake L45, isolated fromnatural fermented sausage as previously described (25), waspropagated at 30°C in MRS broth without Tween 80 (pre-pared from the basic ingredients obtained from Difco Labo-ratories, Detroit, Mich.). Pediococcus acidilactici Pac 1.0,which was used as the indicator strain, was obtained fromM. Daeschel (North Carolina State University, Raleigh), andpropagated in MRS broth (Difco) at 30°C.

Bacteriocin assay. The bacteriocin, lactocin S, was quan-tified in a microtiter plate assay system as previously de-scribed (25). Twofold dilutions of bacteriocin extracts (100,ul) in MRS broth were prepared in microtiter plates (Costar,Cambridge, Mass.). Twenty microliters of fresh indicatorculture (A6. = 0.1 to 0.4) and 80 ,u1 of MRS broth wereadded, and the plates were incubated overnight at 30°C.Growth inhibition of the indicator organism was then mea-sured spectrophotometrically at 600 nm by using a TitertekMultiskan (Flow Laboratories, Helsinki, Finland). One bac-teriocin unit (BU) was defined as the amount of bacteriocinwhich inhibited growth of the indicator organism by 50%(50% of the turbidity of the control culture without bacteri-ocin) under standard assay conditions.Ammonium sulfate precipitation. All the purification steps

were performed at room temperature if not otherwise stated.All the chromatographic equipment was obtained from Phar-macia-LKB Biotechnology (Uppsala, Sweden). MRS brothwithout Tween 80 was inoculated with L. sake L45 (0.2%inoculum) and incubated overnight at 30°C. Overnight cul-tures contained a bacteriocin titer of 300 to 500 BU/ml. The

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TABLE 1. Purification of lactocin S

Vol Total Total activity b Increase YieldPurification stage (ml) A280' (BU) Sp act in sp actb (%)

Culture supernatant 24,000 547,000 11,700,000 21 1 100Fractions

I (Ammonium sulfate precipitation) 900 19,700 11,100,000 560 27 95II (Anion exchange) 1,400 5,000 8,200,000 1,600 76 70III (Cation exchange) 450 1,650 9,200,000 5,600 270 79IV (Phenyl Sepharose) 200 115 4,200,000 36,000 1,700 36V (Gel filtration)' 42 7 1,700,000 240,000 11,000 15VI (Phenyl Superose) 22 1.3 700,000 540,000 26,000 6VII (C2/C18 column) 2 0.35 310,000 900,000 40,000 3a Total A280 equals the optical density at 280 nm multiplied by the volume in milliliters.b Specific activity is bacteriocin units (BU) divided by the optical density at 280 nm.c Prior to gel filtration, fraction IV was transferred to distilled water by gel filtration using PD-10 columns and concentrated to 7 ml by lyophilization.

cells from between 20 and 25 liters of overnight cultureswere removed by centrifugation at 16,000 x g for 10 min at4°C. The supernatant was adjusted to pH 6.5, and ammo-nium sulfate was added to a final concentration of 20%(wt/vol) at 4°C. Lactocin S was pelleted by centrifugation at8,000 x g for 20 min and dissolved in 20 mM sodiumphosphate buffer, pH 7.5 (375 ml per 10-liter culture) (frac-tion I).

Ion-exchange chromatography. Traces of ammonium sul-fate in fraction I were removed by passing it throughSephadex G-25 PD-10 columns equilibrated with 20 mMsodium phosphate, pH 7.5. The lactocin S preparation wassubsequently applied to an 80 ml Q-Sepharose anion-ex-change column equilibrated with the phosphate buffer. Theflowthrough fraction (fraction II), containing lactocin Sactivity, was adjusted to pH 5.2 and applied to an 80-mlS-Sepharose cation-exchange column equilibrated with 20mM sodium phosphate, pH 5.2. The activity was eluted withthe same buffer containing 1.0 M NaCl (fraction III).Hydrophobic interaction and reverse-phase chromatogra-

phy and gel filtration. Ammonium sulfate was added tofraction III to a final concentration of 6% (wt/vol) and wassubsequently applied to a Phenyl Sepharose CL-4B column(18-ml column volume per 6-liter culture) equilibrated with20 mM sodium phosphate, pH 5.2, and 6% (wt/vol) ammo-nium sulfate. The activity was eluted from the column bygradually decreasing the ammonium sulfate concentration(total gradient volume, 136 ml). The flow rate was 4.0ml/min, and 8-ml fractions were collected. The lactocinS-containing fractions (fraction IV) were transferred to dis-tilled water by gel filtration using the PD-10 columnsas described above, concentrated by lyophilization, dis-solved in 7 ml of 20 mM sodium phosphate (pH 6.9)-0.1 MNaCl, and finally applied to a Sephacryl S-100 HR column(2.6 by 90 cm) equilibrated with 0.1 M NaCl in 20 mMsodium phosphate, pH 6.9. Six-milliliter fractions werecollected, and the bacteriocin-containing fractions werepooled (fraction V). The column was calibrated with bovineserum albumin (molecular weight, 67,000), ovalbumin (molec-ular weight, 43,000), chymotrypsinogen (molecular weight,25,000), and RNase A (molecular weight, 13,700). Ammo-nium sulfate was added to fraction V to a final concentrationof6% (wt/vol), followed by application to a Phenyl SuperoseHR 5/5 column, equilibrated with 20 mM sodium phosphate,pH 6.9, and 6% (wt/vol) ammonium sulfate. The flow ratewas 0.8 ml/min, and the lactocin S activity was eluted bygradually decreasing the ammonium sulfate concentration.Fractions containing lactocin S activity (fraction VI) were

directly chromatographed on a C2/C18 reverse-phase col-umn, PepRPC HR 5/5, equilibrated with 5 mM sodiumphosphate, pH 6.9. The flow rate was 1 ml/min, and lactocinS activity was eluted with a linear methanol gradient (totalvolume of 30 ml). Hydrophobic interaction, reverse-phasechromatography, and gel filtration were performed with theuse of the FPLC system (Pharmacia-LKB Biotechnology,Uppsala, Sweden).Amino acid composition and sequence analysis. After chro-

matography on the C2/C18 column, purified lactocin S wasconcentrated in a SpeedVac Concentrator (Savant Instru-ments Inc., Farmingdale, N.Y.). For determination of theamino acid composition, the polypeptides were hydrolyzedin 6 N HCl (or in 6 N HCl containing 3% [vol/vol] thioglycol-lic acid for quantitation of tryptophan) under vacuum at110°C for 24 h (22). The half-cystine content was determinedas cysteic acid after performic acid oxidation (11). Theamino acid analysis was performed on a Biotronik LC5000analyzer, which was connected to a computing integratorsystem (SP 4100 Computing Integrator; Spectra PhysicsInc., San Jose, Calif.). The polypeptides were cleaved withcyanogen bromide as described by Sletten and Husby (31).The amino acid sequence was determined by Edman degra-dation using an Applied Biosystems 477A automatic se-quence analyzer with an on-line 120A phenylthiohydantoinamino acid analyzer (5).The hydropathic index of lactocin S was determined by

using the SOAP program ofPC Gene (IntelliGenetics, Moun-tain View, Calif.) (19, 20). The sequence was compared withthe content of the SWISS-PROT data base by using theDNASIS sequence program package (Pharmacia-LKB) on aVAX/780 computer with the University of Wisconsin Genet-ics Computer Group program package (8).

RESULTS

Purification of lactocin S. Lactocin S, which is found in themedia in the late-exponential phase of growth (24), wasconcentrated approximately 25-fold from the culture mediaby ammonium sulfate precipitation (fraction I). This resultedin a 25- to 30-fold increase in the specific activity with arecovery of approximately 95% of the activity (Table 1,fraction I). After fraction I was passed through an anion-exchange column, followed by binding to a cation ex-changer, the specific activity of lactocin S increased by 250-to 300-fold and the recovery was 70 to 80% (Table 1, fractionIII). Upon subsequent hydrophobic interaction chromatog-raphy on Phenyl Sepharose, lactocin S eluted between 1.2

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AMINO ACID SEQUENCE OF LACTOCIN S 1831

0.550

0.500

0.450

0.400

0.350

0 0.300co< 0.250

0.200

0.150

0.100

0.050

78 K726660I54 ,48 g42 Dm36 >

30 .>6-C.)3024 <1812

6

25 30 35 40 45 50 55 60 65 70Fraction

FIG. 1. Gel filtration on a Sephacryl S-100 column of fraction IV of lactocin S. The bar indicates which fractions were collected for furtherpurification. The amount applied to the column represents that obtained from an approximately 24-liter culture. Six-milliliter fractions werecollected.

and 0% (wtlvol) ammonium sulfate (data not shown). Thespecific activity was at this stage about 1,700-fold higherthan that of the culture supernatant, and the recovery was36% (Table 1, fraction IV).Upon gel filtration of fraction IV, the activity eluted as a

peak with a molecular weight less than that of the smallestprotein standard, RNase A (molecular weight, 13,700) (Fig.1). At this stage, a shoulder on the main, broad absorbancepeak coincided with the peak of lactocin S activity (Fig. 1).After gel filtration, the specific activity had increased ap-proximately 11,000-fold and the recovery was about 15%(Table 1, fraction V). Lactocin S was then subjected tohydrophobic interaction chromatography on a Phenyl Super-ose column, and it eluted between 3.5 and 3.0% ammoniumsulfate (data not shown). A distinct absorbance peak coin-cided with the activity peak. The specific activity hadincreased about 26,000-fold, and the recovery was 6% (Table1, fraction VI). The final purification step, resulting in purelactocin S, was reverse-phase chromatography. The activitypeak, which was recovered in one fraction at a methanolconcentration of approximately 87% (vol/vol), coincidedcompletely with the absorbance peak (Fig. 2). The finalspecific activity of pure lactocin S was 40,000-fold greaterthan in the culture supernatant, and the recovery was 3%(Table 1, fraction VII).Amino acid composition and sequence analysis. The amino

acid composition of lactocin S obtained after the finalreverse-phase chromatography step (fraction VII) was deter-mined (Table 2). Calculation of the number of probableamino acid residues in the bacteriocin molecule revealed thatlactocin S most likely contains 33 amino acids. No tryp-tophan residues were found upon specific analysis for thepresence of this amino acid. The amino acid analysis re-sulted in one extra unknown peak eluting between prolineand glycine which disappeared after performic acid oxida-tion of lactocin S (data not presented). Performic acid-

oxidized material revealed two residues of cysteic acid permolecule. This suggests that two modified cysteine residuesare present in the bacteriocin molecule.Amino acids were not detected upon direct N-terminal

amino acid sequencing of lactocin S, indicating that theN-terminal amino acid was blocked. According to the aminoacid composition, one methionine was present in lactocin S.Lactocin S was therefore subjected to cleavage with cyano-gen bromide, which is specific at methionine residues.Amino acid sequencing was performed on the cleaved prod-uct. The sequencing revealed only one sequence, whichmeans that the N-terminal blocking still was present. Fromthe C-terminal part of the lactocin S molecule 25 amino acidswere obtained, including the cleaved methionine and 3amino acid residues which could not be identified. Thesequence was Met-Glu-Leu-Leu-Pro-Thr-Ala-Ala-Val-Leu-Tyr-Xaa-Asp-Val-Ala-Gly-Xaa-Phe-Lys-Tyr-Xaa-Ala-Lys-His-His, where Xaa represents unidentified residues (Fig.3A). The hydropathic index of lactocin S as determined fromthe known amino acid sequence was 1.37, indicating ahydrophobic nature (20).

DISCUSSION

This is the first report on the purification to homogeneityof a bacteriocin from a facultative heterofermentative Lac-tobacillus ("Streptobacterium") sp. The purification of thebacteriocin-lactocin S, produced by L. sake-resulted in a40,000-fold increase in the specific activity and enabled thedetermination of a major part of its amino acid sequence.

Initial work on concentrating the bacteriocin from regularMRS culture medium was not reproducible, and the recov-ery was often not more than 20%. Various amounts oflactocin S activity were found in the foam and in a floatinglipidlike material after the ammonium sulfate precipitate waspelleted; this activity might be due to the hydrophobic

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-* H#-o

-p-p0

-p0

-p

-p~~~~~~~A..-p

/

/I

//

//

/

5

K

275

250

-225

200

175

150

125

100

75

50

25

I-I

E

m

-0

10Fraction

FIG. 2. Reverse-phase chromatography on a C2/C58 column of fraction VI of lactocin S. The amount applied to the column represents thatobtained from an approximately 16-liter culture. No activity was detected in the flowthrough. The gradient volume was 25 ml, and thebacteriocin was collected in one 1-ml fraction.

character of lactocin S. Lactacin F produced by L. acido-philus has also been reported to be lost in the floatingfraction upon ammonium sulfate precipitation, and thisbacteriocin was found to be associated with lipidlike material(26a). Exclusion of the nonionic detergent, Tween 80, fromthe MRS broth resulted in a high, reproducible recovery oflactocin S in the ammonium sulfate precipitate.The apparent molecular weight of lactocin S as determined

by gel filtration varied. After ammonium sulfate precipitationfrom the culture medium, lactocin S has previously beenshown to elute with a molecular weight of about 30,000 (24),whereas in a more purified state it always eluted at amolecular weight of less than 13,700 (RNase A). It appearedthat the more crude the bacteriocin was during gel filtration,

TABLE 2. Amino acid composition

Amino acid Residues permolecule"

Ala.7.60 (7-8)Asp.0.92(1)Glu.1.06 (1)Gly.1.10 (1)His.2.03(2)Leu.3.80(4)Lys.2.09(2)Met.1.05(1)Phe.1.04 (1)Pro.1.9(2)Thr.0.90 (1)Tyr.1.84 (2)Val.4.5 (4-5)Cysb............................... 1.8 (2)

the higher the apparent molecular weight, suggesting that itassociates with other macromolecules. Its behavior upon gelfiltration in a partially purified state (Fig. 1) indicates that itperhaps also associates with itself and forms dimers ortrimers. Other bacteriocins produced by lactobacilli alsoaggregate or associate with other substances under nondis-sociating conditions. Upon gel filtration, lactocin 27 eluteswith a molecular weight of more than 200,000, whereasunder dissociating conditions a glycoprotein with an appar-ent molecular weight of 12,400 exhibits bacteriocin activity(33). Ultrafiltration experiments of crude extracts of lactacinB suggested a molecular weight of 100,000 (1). However,further analysis of a more pure bacteriocin preparationrevealed a molecular weight of 6,500 (2). Also, helveticin Jwas present as an aggregate with a molecular weight above300,000. Upon purification and sodium dodecyl sulfate-gelelectrophoresis, however, the bacteriocin migrated as a37,000-Da protein that retained the inhibitory activity (14).The amino acid composition indicated that lactocin S

consists of 33 amino acids, of which about 50% are thenonpolar amino acids alanine, valine, and leucine (Table 2).The hydropathic index (20) was calculated from the se-quenced part of the molecule to be 1.37, and the hydropho-bic character of the molecule is consistent with its relativelyhigh affinity to the phenyl and C2/C18 columns. The 25-residue C-terminal part of lactocin S which was sequencedcontained three amino acid residues that were not identified.It is likely that the unidentified residues are modified formsof cysteine and/or amino acids associated with cysteine in amanner similar to that seen in lanthionine residues present innisin, since two cysteic acids per lactocin S molecule werefound upon performic acid oxidation of lactocin S. In the Nterminus, lactocin S consists of approximately 8 amino acidsof unknown sequence. From the amino acid composition,this sequence is highly nonpolar, consisting mainly of 3 to 4alanine and 2 to 3 valine residues.

1.10

1.00

0.90

0.80

0.70

0.60

0.50

0.40

0.30

I-I

CM

C\j

0.20

0.10

" Mean values from three runs. The values in parentheses are the numbersof the individual amino acids in lactocin S.

b Determined as cysteic acid after performic acid oxidation of the polypep-tide.

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AMINO ACID SEQUENCE OF LACTOCIN S 1833

SEQUENCE FOR

1 M ELL PTAAVLYXDVAGXFKYXAKHH 25 part of lactocin S

0O

m

C)

2 ELa) I I

41 EL

L PTAAVLYXDVAGXFKYXAKHH 25ml ii,l l l lml ii.l l l l

L PTAAVSRRDPATPAEPVVRL D 64

part of lactocin S

Flavobacterium6-aminohexanoate-dimer hydrolase

3 LLPTAAVLYXDVAGXFKYXAKI lI I II I IlI I I I I I l

4 LLPTAAAGLLLLAAQPAMAAN

1 MELLPTA**AVLYXDVAGXFKYm ml I II l I

I lI I I I Il

2 LELLPTAVEGVSQAQ ITGRPEW

23 part of lactocin S

24 Erwinia carotovorapectate lyase bprecursor

20

23

part of lactocin S

Halobacterium halobiumbacterio-rhodopsinprecursor

FIG. 3. (A) Amino acid sequence of the C-terminal part of lactocin S. (B) Comparison of lactocin S amino acid sequence with homologoussequences found in three bacterial proteins.

Nisin is the only bacteriocin from lactic acid bacteria forwhich the primary amino acid structure has been published.Lactocin S does not share any homology to nisin. A newlactococcal bacteriocin consisting of 54 amino acids has beenisolated and sequenced (13). This lactococcal bacteriocinalso contains two histidine residues at the very C-terminalend of the molecule, but no other homology to lactocin S wasfound (13). Van Belkum et al. published the sequences oftwo bacteriocins from L. lactis subsp. cremoris. Theselactococcal bacteriocins differ from lactocin S, but one ofthese may also contain two histidine residues at the veryC-terminal end of the molecule (34). Since this work wassubmitted, Muriana and Klaenhammer (28) published thesequence of 25 consecutive N-terminal amino acids of lact-acin F (27) and the nucleotide sequence of this bacteriocingene (28). Lactacin F is certainly different from lactocin S.The known amino acid sequence of lactocin S was unique

when compared to the SWISS-PROT data bank. Threeproteins, however, revealed partial homology with lactocinS: the pectate lyase B precursor isolated from Erwiniacarotovora (21), the bacteriorhodopsin precursor isolatedfrom Halobacterium halobium (17), and the 6-aminohex-anoate-dimer hydrolase from Flavobacterium sp. strainK172 (29) (Fig. 3B). Two of these sequences, the pectatelyase B precursor and the bacteriorhodopsin precursor, are

part of a signal sequence.The hydrophobic nature of lactocin S and its homology

with signal sequences suggest the cell membrane as a possi-

ble target for lactocin S. Because of the potential use oflactic bacteriocins, it is of great importance to gain insightinto their genetics, chemical nature, and mode of action.This study presents biochemical characteristics and a majorpart of the amino acid sequence of a new bacteriocin foundin L. sake, information which is of importance for elucidat-ing the genetics and mode of action of this bacteriocin.

ACKNOWLEDGMENTS

This study was supported by both a fellowship to C.I.M. and a

research grant from the Agricultural Research Council of Norway.

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